We have isolated and characterized regions important for expression of the mouse Na+/H+ exchanger gene. A 1.1-kilobase fragment upstream of the 5'-untranslated region contains specific DNA motifs characteristic of promoter and enhancer elements including a TATA box, two CAAT boxes, an SP-1 site, a cyclic AMP response element-binding site, and an AP-2-like site. This 1.1-kilobase fragment directs transcription of a luciferase reporter gene in mouse fibroblasts (NIH 3T3) and human Hep G2 cells. Deletion or mutation of an AP-2-like site 100 base pairs from the start site of transcription resulted in loss of most of the reporter plasmid activity. In addition, cotransfection of an AP-2 expression plasmid and the mouse promoter/luciferase plasmid increased the amount of Na+/H+ exchanger-directed transcription in AP-2-deficient Hep G2 cells. Moreover, mobility shift analysis indicated that a putative AP-2-binding site is capable of binding purified AP-2 protein and a specific protein from nuclear extracts of NIH 3T3 cells. The results show that the transcription factor AP-2 may play an important role in regulation of transcription of the mouse Na+/H+ exchanger gene.

We examined the expression and activity of the Na+/H+ exchanger in the human choriocarcinoma BeWo cell line. When treated with methotrexate, these cells differentiated from cytotrophoblast-like cells to enlarged multinucleate syncytiotrophoblast-like cells. There was no change in the apparent Km for Na+ between undifferentiated and differentiated cells. However, differentiated cells could transport more than five times the proton flux of undifferentiated cells. There was no difference in the Hill coefficient between undifferentiated and differentiated cells. However, the maximal flux (Jmax) for undifferentiated cells was higher than that for differentiated cells. Inhibition of Na+/H+ exchange activity by an amiloride analog and Hoe694 revealed a sensitive and a resistant component in both differentiated and undifferentiated cells. Northern blot analysis and immunocytochemistry suggested that the sensitive component was due to the NHE1 isoform of the protein while the resistant component was due to the NHE3 isoform. The NHE1 isoform was localized to the brush border membrane of BeWo cells and Western blot analysis showed that the NHE1 protein was more abundant in brush border membranes from differentiated BeWo cells compared to undifferentiated cells. The results show that BeWo cells contain the NHE1 and NHE3 isoforms of the Na+/H+ exchanger and that the NHE1 isoform is primarily localized to the brush border membrane.

No abstract